PROJECT SUMMARY/ABSTRACT Humanized mice, i.e., immunodeficient mice that support the development of human immune systems by engraftment with human hematopoietic stem cells (HSCs) have become increasingly important preclinical tools. However, HSC engraftment of NSG mice results in the development of H2-restricted human T cells that cannot interact appropriately with autologous human HLA-expressing, antigen-presenting cells and B cells in the periphery. NSG-HLA transgenic mice have been generated to address this, but the repertoire of HLA specificities is restricted to only those expressed and H2-restricted human T cells continue to be generated in the murine thymus. The most robust immune system in humanized NSG mice is created using fetal tissues, the BLT (bone marrow/liver/thymus) model. Use of the BLT model permits human T cell development on an autologous thymus and generates robust HLA-restricted human immune systems. However, some human T cells still develop in the murine thymus and are H2-restricted, confounding interpretation of the experiments. In addition, the use of human fetal tissue to create humanized mice is complicated by its limited availability and by concerns surrounding procurement of the tissues. To provide an alternative to fetal tissue-based models, we propose to utilize novel athymic NSG mouse strains developed by crosses of human cytokine trangenic NSG mice with our athymic NSG-Foxn1null (nude) mouse stock and by CRISPR/Cas targeting Foxn1 directly in our new NSG stocks expressing human hematopoietic growth factors. We will use umbilical cord blood-derived iPS cells to create human thymus structures that will support in vivo development of HLA-restricted human T cells derived from autologous cord blood HSCs. For this multi-PI, multi-disciplinary team project, we propose three Aims: 1) Generate and validate new models of immunodeficient mice that support optimal engraftment of HSCs and function of human thymic tissues in the absence of murine thymus; 2) Generate and validate human thymic epithelium and organoids generated from iPS cells; 3) Compare the human immune system generated in our new models with the BLT model. Our proposal takes advantage of powerful new technologies for creating new models of humanized mice, and builds on our >25 year track record for generating, validating, and sharing novel models of humanized mice. We believe that our innovative approaches, combined with our multi-disciplinary collaborative team will ensure the development of this much-needed preclinical model of humanized mice that does not rely on use of human fetal tissues. Furthermore, Dr. Shultz is at The Jackson Laboratory where these mouse models will be developed, facilitating their rapid distribution to the scientific community and uniquely positioning us to achieve the stated goals of this program announcement.